Sales of the serine protease subtilisin exceed $ 300 million annually, accounting for approximately 40% of the industrial enzyme market. For more than 30 years, proteases, including subtilisin, have been used as additives in laundry and other detergents. Subtilisin has a broad specificity for proteins that commonly soil clothing, including proteins found in blood, grass, soil and many food products.
Initially isolated from the bacteria Bacillus subtilis, subtilisin has become one of the most intensively studied and extensively engineered proteins known to date. A wide variety of subtilisins have been identified, and the amino acid sequences of a number of these subtilisins have been determined. In addition, structural investigations, including more than 100 crystal structures, have revealed that subtilisins share a common active site with other serine proteases, the Ser-His-Asp catalytic triad.
Despite such studies, structural features correlating with specific functional properties remain to be elucidated. Indeed, due both to the lack of structural predictability and to the need to optimize multiple characteristics simultaneously, the task of protein engineering remains difficult.
For example, in detergent applications, subtilisins are not only active under a variety of washing conditions, they are also stable in the presence of other detergent components and additives. Such additives may include, among other things, other enzymes such as cellulases, lipases and the like. Subtilisin should be stable in the presence of effective concentrations of such enzymes, and at the same time must not result in the degradation (proteolysis) of these enzymes. The subtilisin selected for such an application should also be active under a variety of specific conditions such as high or low temperature, acid, neutral or alkaline pH, or the presence of such additives as bleaching agents. Mutations or alterations in the nucleotide or amino acid sequences which would provide these benefits are difficult to predict, and therefore difficult to engineer.
Nonetheless, both random mutagenesis and targeted mutagenesis approaches have been applied to the goal of producing improved subtilisin homologues. However, attempts to develop proteases that are improved for multiple properties are hampered by the fact that random mutations are often deleterious, and attempts to rationally alter one property of an enzyme often disrupt other important existing characteristics (Patkar et al. (1998) Chem Phys Lipids 93:95; Shoichet et al. (1995) Proc Natl Acad Sci USA, (1995) 92:452).
The present invention provides novel subtilisin homologues that are improved for a variety of specific properties including thermal stability, activity at low temperature, alkaline stability as well as other desirable properties and combinations of properties. These subtilisins are useful in a variety of detergent and other industrial and commercial applications.